著者

Yusuke Suzuki Atsushi Kouzuma Kazuya Watanabe

出版者

Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

雑誌

The Journal of General and Applied Microbiology (ISSN:00221260)

巻号頁・発行日

pp.2019.04.007, (Released:2019-08-23)

参考文献数

26

被引用文献数

6

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Here, we developed an all-in-one, broad host-range CRISPR/Cas9 vector system widely applicable to genome editing of proteobacteria. Plasmid pBBR1-Cas9 was constructed by cloning the cas9 gene from Streptococcus pyogenes into the broad host-range plasmid pBBR1MCS-2. We evaluated its applicability for frameshift mutagenesis of Shewanella oneidensis MR-1. Significant cell death was observed when MR-1 cells were transformed with a pBBR1-Cas9 derivative that expressed a single-guide RNA targeting the crp gene. However, cell death was partially prevented when a donor DNA fragment containing a modified crp sequence with a frameshift mutation was introduced using the same vector. All transformants (9 colonies) contained the expected frameshift mutation in their chromosomal crp genes. These results indicate that this vector system efficiently introduced CRISPR/Cas9-mediated double-strand DNA breaks and subsequent homology-directed repair. This work provides a simple and powerful genome-editing tool for proteobacteria that can harbor pBBR1-based plasmids.